Direct nucleic acid analysis

ABSTRACT

Methods and apparatus are described for nucleic acid analysis of swab samples without the need for purification.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. provisional patentapplication Nos. 61/319,610, filed Mar. 31, 2010, 61/357,043, filed Jun.21, 2010, 61/414,776, filed Nov. 17, 2010, and 61/414,772, filed Nov.17, 2010, all of which are hereby incorporated by reference herein intheir entireties.

TECHNICAL FIELD OF THE INVENTION

The invention relates to the field of diagnostic assays, in particular,nucleic acid amplification-based assays.

BACKGROUND

Nucleic acids are used frequently in the clinical setting to identifygenetic mutations and to diagnose bacterial and/or viral infections.Such methods generally require use of nucleic acids isolated orextracted from biological samples.

A commonly used method for isolating DNA from a biological sample (e.g.,blood or saliva) involves lysing the cells contained in the sample witha combination of a proteolytic enzyme and a detergent followed byextracting the mixture with an organic solvent, e.g., phenol andchloroform, so that the DNA enters the aqueous phase and the proteinenters the organic phase. The DNA in the aqueous phase is thenconcentrated by alcohol precipitation and re-suspended in a suitablevolume for analysis. Such methods are, however, time-consuming andgenerally require the use of toxic reagents.

Simpler methods using fewer reagents have been reported. For example,DNAzol™ Direct (Molecular Research Center, Inc.) is an alkaline solutioncontaining polyethylene glycol and other additives. Buccal swabs andwhole saliva samples are incubated in DNAzol™ Direct for 15 min. Theresulting lysate may be added directly to a polymerase chain reaction(PCR) mix, where the lysate should be less than 10% of the PCR mix byvolume.

In another example, 0.25 μl to 5 μl of whole saliva or buccal swabsample may be added directly into a 50 μl PCR mixture containing 1×EzWay™ Direct PCR Buffer (Koma Biotech). The PCR cycling program shoulduse an initial denaturation of 95° C. for 5 min followed by cyclingdenaturation steps of 94° C. for 30 sec to 60 sec.

The disadvantages of DNAzol™ Direct and EzWay™ Direct PCR Buffer arethat they require the use of special reagents and specific incubation ordenaturation conditions.

Another approach to bypassing the DNA purification step is the use ofspecially-modified DNA polymerase enzymes that are resistant toinhibitors in samples. For example, the Phusion™ Human Specimen DirectPCR Kit (Finnzymes) claims to allow PCR directly from unpurified humansamples. A tiny amount of sample is used directly in the PCR reactionwith no prior purification steps. Suitable sample materials includebuccal swabs, saliva, amniotic fluid, hair, fingernails, teeth, and skinbiopsies. The kit is based on modified Phusion™ Hot Start IIHigh-Fidelity DNA Polymerase. This specially engineered proofreading DNApolymerase is fast, robust and highly tolerant of many PCR inhibitorspresent in human tissues. The disadvantage of this approach is that itrequires a proprietary DNA polymerase that may be more expensive thanstandard DNA polymerase.

Simpler methods for analyzing whole saliva samples have been reported.For example, Ochert et al. (1994) boiled whole saliva for 5 min and thenused it as the sample for PCR. This method resulted in successful PCRfor 7 out of 10 samples. The researchers used 30 μl of boiled sample ina total reaction volume of 50 μl. The PCR cycling program used cyclingdenaturation steps of 1 min at 94° C. One of the samples, which gave aninconclusive result with 5 min of boiling, generated a positive resultafter 6 or more minutes of boiling. The researchers concluded that:“[t]he chemical nature of the inhibitors remains to be characterized. Byinference, they are unlikely to be proteins because of their relativeheat resistance and the observation that extraction procedures based onphenol-chloroform or proteinase K did not prevent inhibition. Thepersistence of inhibition after processing with gel matrices orion-exchange resins suggests that low-molecular-weight ionic moietiesare also not inhibitors. Polysaccharides are possible candidates.”(Ochert A S et al. (1994) Inhibitory effect of salivary fluids on PCR:potency and removal. Genome Research. 3: 365-368.)

In another example, French et al. (2002) performed direct PCRamplification from whole saliva using HyBeacon™ probes on theLightCycler™ instrument. PCR volumes were typically 20 μl, containing 2μl of saliva. Saliva samples were diluted to 50% in water. The PCRmixtures used 1 unit of Taq DNA polymerase (Taq from Amersham PharmaciaBiotech or Z-Taq from TaKaRa). The final concentration of PCR primerswas 0.5 μM each, and the final concentration of HyBeacon™ fluorescentprobe was 150 nM. The PCR cycling program used an initial denaturationstep of 95° C. for 5 min followed by cycling denaturation steps of 95°C. for 0 seconds (i.e., the thermal cycler ramped up to 95° C. andimmediately ramped down to the extension temperature). Saliva sampleswere typed on the same day that they were collected, although asignificant reduction in assay efficiency was not observed with salivasamples stored at 4° C. for 2-3 days, or −20° C. The researchers notedthat since the number of buccal epithelial cells shed into salivasamples may not be equal from person-to-person and day-to-day, a furtherstudy of assay reproducibility and robustness may be required. (French DJ et al. (2002). Ultra-rapid DNA analysis using HyBeacon™ probes anddirect PCR amplification from saliva. Molecular and Cellular Probes. 16:319-326.)

Researchers have found that there is high variability in theconcentration of DNA from saliva or buccal swabs in a population ofsubjects. For example, Garcia-Closas et al. (2001) collected twocytobrush samples each from 40 individuals and found that the amount ofhuman DNA ranged from 0.006 to 13.5 μg after phenol-chloroform DNApurification (Garcia-Closas M et al. (2001). Collection of Genomic DNAfrom Adults in Epidemiological Studies by Buccal Cytobrush andMouthwash. Cancer Epidemiology, Biomarkers & Prevention. 10(6):687-696.).

Although boiling whole saliva for 5 minutes or more, or diluting wholesaliva and then denaturing at 95° C. for 5 min are relatively simplesteps, they still require time and effort on the part of the user. Forautomated instruments, these steps would require extra mechanical partsand movements to accomplish. In addition, the PCR success rate of 7 outof 10 boiled saliva samples reported by Ochert et al. (1994) makes thismethod less desirable for clinical applications where higher reliabilityis typically expected.

SUMMARY OF THE INVENTION

The present disclosure provides methods, systems, and apparatuses forcollecting and/or amplifying nucleic acids. In one aspect, the presentdisclosure provides methods that involve contacting a sample including anucleic acid with a nucleic acid amplification reagent withoutpurification of nucleic acids from the sample. In some embodiments, thenucleic acid amplification reagent contains at least one of a DNApolymerase at a concentration of at least 1.0 U/reaction, a primer at aconcentration of at least 0.2 μM, and a probe of at least 0.2 μM. Thesample may, for example, be a swab sample and may be a buccal swabsample. In some embodiments, the DNA polymerase utilized is notspecially modified to resist inhibitors. In some embodiments, the DNApolymerase is no more resistant to inhibitors in human samples than is areference DNA polymerase such as, for example Taq DNA polymerase. Insome embodiments, the concentration of DNA polymerase is 2.0 U/reactionor higher.

In some embodiments, the nucleic acid amplification reagents containprimer and probe concentrations higher than typically used in PCRreactions (0.1-0.2 μM). In some embodiments, the primer concentration isgreater than 0.2 μM and the probe concentration is greater than 0.2 μM.In some embodiments, the primer concentration is 0.5 μM and the probeconcentration is 0.7 μM.

In some embodiments, the nucleic acid amplification reagents containboth DNA polymerase and primer and probe concentrations higher thantypically used in PCR reactions.

In some embodiments, the methods further comprise determining whether anamplification product was produced as a result of the nucleic acidamplification reaction.

In some embodiments, the nucleic acid amplification reaction isperformed in a reaction vessel and a cap of the reaction vessel is usedto collect the buccal sample (e.g., by contacting the cap with theinside cheek or tongue of the subject).

In some embodiments, the cap of the reaction vessel is incorporated intoa cap holder device so that the device functions as a buccal swab.

In some embodiments, the nucleic acid amplification reaction isperformed within 120 minutes of contacting the buccal sample with thenucleic acid amplification reagents. In some embodiments, the nucleicacid amplification reaction is performed within 60, 30, 15, 10, 5, or 1minutes of contacting the buccal sample with the nucleic acidamplification reagents.

In some embodiments, the nucleic acid amplification reaction comprisesan initial heat denaturation step of 15 minutes or less. In someembodiments, the nucleic acid amplification reaction comprises aninitial heat denaturation step of 5 minutes or less, 3 minutes or less,or 1 minute or less.

In some embodiments, the swab sample is collected from a mammal (e.g., ahuman, dog, cat, cow, sheep, pig, etc.). In some embodiments, the swabsample is collected from an open body cavity. In some embodiments, theswab sample is collected from a body surface. In some embodiments, theswab sample is a buccal sample. In some embodiments, the swab sample iscollected from the palm of a hand, inside the folds of the pinna of anear, an armpit, or inside a nasal cavity.

In some embodiments, the swab sample is collected from a foodstuff. Insome embodiments, the foodstuff is raw. In some embodiments, thefoodstuff is a fruit, a vegetable, a meat, a fish, or a shellfish. Insome embodiments, the meat is pork, beef, chicken, or lamb.

In some embodiments, the nucleic acid amplification reaction isperformed in a reaction vessel having a removable cap and the cap isused to collect the swab sample. In some embodiments, a means forholding the cap of the reaction vessel is used when collecting the swabsample. In some embodiments, the cap is contacted with the inside of acheek or a tongue. In some embodiments, the cap is contacted with thepalm of a hand, the inside of the folds of the pinna of an ear, anarmpit, or the inside of a nasal cavity. In some embodiments, the cap iscontacted with a foodstuff.

In some embodiments, the swab sample is collected from a source and themethod is repeated with at least one other swab sample from the samesource.

In some embodiments, the swab sample is directly contacted with thenucleic acid amplification reagents without any intervening steps. Insome of these embodiments, the nucleic acid amplification reaction isperformed in a reaction vessel having a removable cap and the cap isused to collect the swab sample. In some embodiments, the reactionvessel comprises the nucleic acid amplification reagents and the cap isused to seal the reaction vessel after the swab sample has beencollected. In some embodiments, a means for holding the cap of thereaction vessel is used when collecting the swab sample.

In some embodiments, the kit further comprises nucleic acidamplification reagents. In some embodiments, the cap is mated with thereaction vessel and the nucleic acid amplification reagents are locatedwithin the reaction vessel. In some embodiments, the reaction vessel isempty and the nucleic acid amplification reagents are located within oneor more containers that form part of the kit.

In some embodiments, the cap is mated with the reaction vessel.

In some embodiments, the cap is mated with the means for holding thecap.

In some embodiments, the kit further comprises a means for controlling anucleic acid amplification reaction in the reaction vessel. In someembodiments, the means for controlling a nucleic acid amplificationreaction in the reaction vessel comprises a thermal cycler with a wellfor receiving the reaction vessel.

In some embodiments, the kit further comprises a means for determiningwhether an amplification product has been produced as a result of thenucleic acid amplification reaction. In some embodiments, the means fordetermining whether an amplification product has been produced as aresult of the nucleic acid amplification reaction comprises a device formeasuring fluorescence in the reaction vessel while the reaction vesselis located within the means for controlling a nucleic acid amplificationreaction in the reaction vessel.

In another aspect, the methods involve contacting non-buccal samplesfrom human and non-human sources with nucleic acid amplificationreagents without purification of nucleic acids in the non-buccal sampleand then performing a nucleic acid amplification reaction on a nucleicacid template in the non-buccal sample. In some embodiments, the methodsfurther comprise determining whether an amplification product wasproduced as a result of the nucleic acid amplification reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

Certain features of a provided closure system are described in detailherein below with reference to the figures, wherein:

FIG. 1 shows a cross-sectional view of a Spartan DX-12 tube with the capfully inserted in accordance with embodiments of the present invention.

FIG. 2 shows front, bottom, and perspective views of a Spartan DX-12tube of FIG. 1 with the cap fully inserted.

FIG. 3 shows side and top views of a Spartan DX-12 tube of FIG. 1 withthe cap fully inserted.

FIG. 4 shows perspective views of a Spartan DX-12 tube of FIG. 1 withthe cap removed from the tube.

FIG. 5 shows how a cap from a Spartan DX-12 tube may be assembled withan exemplary means for holding the cap in accordance with embodiments ofthe present invention.

FIG. 6 shows how the cap and means for holding the cap shown in FIG. 5are mated.

FIG. 7 shows a graph demonstrating the effect that the concentration ofTaq DNA polymerase has on the Endpoint Florescence (EPF) of buccalsamples amplified by a FAM probe in accordance with embodiments of thepresent invention.

FIG. 8 shows a graph demonstrating the effect that the concentration ofTaq DNA polymerase has on the Endpoint Florescence (EPF) of buccalsamples amplified by a CalFluor 610 probe in accordance with embodimentsof the present invention.

FIG. 9 shows a graph demonstrating the effect that the concentrations ofprimer and probe have on the Endpoint Florescence (EPF) of buccalsamples amplified by a FAM probe in accordance with embodiments of thepresent invention.

FIG. 10 shows a graph demonstrating the effect that the concentrationsof primer and probe have on the Endpoint Florescence (EPF) of buccalsamples amplified by a CalFluor 610 probe in accordance with embodimentsof the present invention.

FIG. 11 shows a graph demonstrating the combined effect that theconcentrations of Taq DNA polymerase, primer, and probe have on theEndpoint Florescence (EPF) of buccal samples amplified by a FAM probe inaccordance with embodiments of the present invention.

FIG. 12 shows a graph demonstrating the combined effect that theconcentrations of Taq DNA polymerase, primer, and probe have on theEndpoint Florescence (EPF) of buccal samples amplified by a CalFluor 610probe in accordance with embodiments of the present invention.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Definitions. As used herein the following terms shall have the meaningsindicated, unless indicated otherwise:

As used herein, the term “about” when used in reference to a numericalvalue, means plus or minus 10%.

As used herein, the terms “amplification” or “amplify” refer to methodsknown in the art for copying a target sequence from a template nucleicacid, thereby increasing the number of copies of the target sequence ina sample. Amplification may be exponential or linear. A template nucleicacid may be either DNA or RNA. The target sequences amplified in thismanner form an “amplified region” or “amplicon.” While the exemplarymethods described hereinafter relate to amplification using PCR,numerous other methods are known in the art for amplification of targetnucleic acid sequences (e.g., isothermal methods, rolling circlemethods, etc.). The skilled artisan will understand that these othermethods may be used either in place of, or together with, PCR methods.See, e.g., Saiki, “Amplification of Genomic DNA” in PCR Protocols, Inniset al. (1990). Eds. Academic Press, San Diego, Calif. pp 13-20; Wharamet al. (2001). Nucleic Acids Res. 29(11): E54-E54; Hafner et al. (2001).Biotechniques. 30(4): 852-6, 858, 860 passim. Further amplificationmethods suitable for use with the present methods include, for example,reverse transcription PCR (RT-PCR), ligase chain reaction (LCR),transcription-based amplification system (TAS), nucleic acid sequencebased amplification (NASBA) reaction, self-sustained sequencereplication (3SR), strand displacement amplification (SDA) reaction,boomerang DNA amplification (BDA), Q-beta replication, or isothermalnucleic acid sequence based amplification.

As used herein, the term “forward primer” refers to a primer thathybridizes to the anti-sense strand of dsDNA. A “reverse primer”hybridizes to the sense-strand of dsDNA.

As used herein, the term “genomic DNA” refers to some or all of the DNAfrom the nucleus of a cell. Genomic DNA may be intact or fragmented(e.g., digested with restriction endonucleases by methods known in theart). In some embodiments, genomic DNA may include sequences from all ora portion of a single gene or from multiple genes, sequences from one ormore chromosomes, or sequences from all chromosomes of a cell.

As used herein, the terms “hybridize” and “hybridization” refer to aprocess where two complementary or partially-complementary nucleic acidstrands anneal to each other as a result of Watson-Crick base pairing.Nucleic acid hybridization techniques are well known in the art. See,e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Press, Plainview, N.Y. Those skilledin the art understand how to estimate and adjust the stringency ofhybridization conditions such that sequences having at least a desiredlevel of complementarities will form stable hybrids, while those havinglower complementarities will not. For examples of hybridizationconditions and parameters, see, e.g., Sambrook, et al., 1989, MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press,Plainview, N.Y.; Ausubel, F. M. et al. 1994, Current Protocols inMolecular Biology. John Wiley & Sons, Secaucus, N.J.

As used herein, the term “nucleic acid” refers broadly to genomic DNA,segments of a chromosome, segments or portions of DNA, cDNA, and/or RNA.Nucleic acids may be derived or obtained from an originally isolatednucleic acid sample from any source (e.g., isolated from, purified from,amplified from, cloned from, reverse transcribed from sample DNA orRNA). Nucleic acids include those resident in a biological sample,preferably a cell sample or a cellular body fluid sample.

As used herein, the term “sense strand” means the strand ofdouble-stranded DNA (dsDNA) that includes at least a portion of a codingsequence of a functional protein. “Anti-sense strand” means the strandof dsDNA that is the reverse complement of the sense strand.

As used herein, the term “swab sample” means a sample obtained with acollection tool. The collection tool may include a small piece of cottonor soft porous foam on the end of the tool, but is not required to.

Referring now to the drawing figures, wherein like reference numeralsidentify similar, identical, or corresponding elements, embodiments ofthe direct nucleic acid analysis apparatus and methods of using andproducing the same are described.

In general, the methods of the present disclosure involve contacting aswab sample with nucleic acid amplification reagents withoutpurification of nucleic acids in the swab sample and then performing anucleic acid amplification reaction on a nucleic acid template in theswab sample. In some embodiments, the nucleic acid amplificationreagents contain DNA polymerase at a concentration higher than typicallyused in PCR reactions (1.0 U/reaction); primer and probe concentrationshigher than typically used in PCR reactions (0.1-0.2 μM); or thecombination of the two conditions.

In some embodiments, the methods further comprise determining whether anamplification product was produced as a result of the nucleic acidamplification reaction.

Collection of Samples

In general, the swab sample is collected by contacting a sample sourcewith a physical structure. Any physical structure that collects a swabsample when contacted with the sample source may be used for thispurpose. In some embodiments, the physical structure may comprise anabsorbent material (e.g., cotton). In some embodiments, the physicalstructure may be made of plastic and may collect the swab sample as aresult of friction.

In some embodiments, the swab sample may be collected using a buccalswab such as the EasySwab™ (TrimGen, Cat. No. ES-100). In someembodiments, the nucleic acid amplification reaction is performed in areaction vessel 100, which has a cap 101 (e.g., the cap of a SpartanDX-12 tube as shown in FIGS. 1-4), which cap is used to collect the swabsample. It is to be understood that any cap configuration may be usedfor this purpose. In some embodiments cap 101 is made of a plasticmaterial. In some embodiments, cap 101 has a tip 106 that protrudesinside the reaction vessel 103 as shown in FIGS. 1-4. Tip 106 of cap 101is used to collect the buccal sample. In some embodiments, cap 101 isseparated from the body of the reaction vessel for this collection step,as shown in FIG. 4, while in other embodiments cap 101 remains connectedto the body of reaction vessel 103 via a flexible arm or cord (notshown).

FIGS. 2-4 provide additional view of the cap and reaction vesselillustrated in FIG. 1. FIG. 2 shows front, bottom, and perspective viewsof a Spartan DX-12 tube of FIG. 1 with the cap fully inserted. Asdemonstrated in FIGS. 1-4 the tube body of reaction vessel 103 may havebe tapered, for example at a 5° angle, in accordance with variousembodiments of the present invention. FIG. 3 shows side and top views ofa Spartan DX-12 tube of FIG. 1 with the cap fully inserted. The sideview provided by FIG. 3 illustrates that lip 102 of tube 103 may extendon only a portion of the periphery of the tube. FIG. 4 shows perspectiveviews of a Spartan DX-12 tube of FIG. 1 with the cap removed from thetube. As further demonstrated in FIG. 4, tip 106 of cap 101 may containa series of graduations such that the outer diameters of the tipsubstantially corresponding to the various inner diameters of tube 103.

In some embodiments, a means for holding cap 101 of the reaction vessel103 is used when collecting the swab sample. Any structure that iscapable of holding cap 101 may be used for this purpose. In someembodiments, at least a portion of tip 106 of cap 101 is formed into ashape corresponding to at least a portion of the shape of the innervolume of reaction vessel 103, such that the vessel and tip engage in amating configuration. The end of cap 101 opposing tip 106 is shaped toengage in a mating configuration with means for holding the cap inaccordance with some embodiments of the present invention. Exemplarystructures for holding cap 101 are shown in FIGS. 5-6, as discussedfurther herein.

In some embodiments, the swab sample is collected from a mammal (e.g., ahuman, dog, cat, cow, sheep, pig, etc.). In some embodiments, the mammalis a human. In some embodiments, the swab sample is collected from anopen body cavity (e.g., mouth, nose, throat, ear, rectum, vagina, andwound). In some embodiments, the swab sample is a buccal sample. In someembodiments, the buccal sample may be collected by contacting (e.g.,touching and/or swiping) cap 101 on the inside of a cheek. In someembodiments, the buccal sample may be collected by contacting cap 101with a tongue rather than a cheek. In some embodiments, the swab sampleis collected from a body surface (e.g., skin). In some embodiments, theswab sample is collected from the palm of a hand, inside the folds ofthe pinna of an ear, an armpit, or inside a nasal cavity. Any of theaforementioned samples may be obtained by touching and/or swiping therelevant surface with a physical structure (e.g., a buccal swab or cap101 of reaction vessel 103).

In some embodiments, the swab sample is collected from a foodstuff. Insome embodiments, the foodstuff is raw. In some embodiments, thefoodstuff is a fruit, a vegetable, a meat, a fish, or a shellfish. Insome embodiments, the meat is pork, beef, chicken or lamb. In someembodiments, the swab sample may be collected by touching and/or swipingthe relevant foodstuff.

In general, two or more buccal samples may be collected from the samesource and subsequently processed according to the methods of thepresent disclosure. As discussed in connection with examples providedherein, testing two or more samples from the same source may reduce andeven eliminate situations where no results are obtained for a particularsource. In some embodiments, the two or more samples may be collectedfrom about the same location within the source (e.g., both from theinner cheek or both from the tongue in the case of a buccal sample). Insome embodiments, the samples may be collected from different locations(e.g., one from the cheek and one from the tongue in the case of abuccal sample). In yet other embodiments, one sample may be collectedand split into two aliquots that are then processed separately. In someembodiments, one sample is collected with a reaction vessel cap orbuccal swab and the cap or swab is then sequentially contacted withnucleic acid amplification reagents in separate reaction vessels. Insome embodiments a cap is used for this purpose and used to cap off thesecond reaction vessel (a fresh sterile cap being used to cap off thefirst reaction vessel). In general, it is to be understood that the twoor more samples may be processed in sequence, in parallel or acombination thereof.

In some embodiments, the swab sample may be collected from a subjectusing cap 101 of reaction vessel 103, such as the Spartan DX-12 tube cap(Spartan Bioscience, Ottawa, Canada). It is to be understood that anycap configuration may be used for this purpose. In some embodiments, thecap may be held by a cap holder device such as in the Spartan swab(Spartan Bioscience, Ottawa, Canada).

Amplification of Nucleic Acids

As detailed herein, samples, which may be buccal swab samples, arecollected and contacted with nucleic acid amplification reagents withoutpurification of nucleic acids in the sample. In certain embodiments, theterm “without purification” means that the nucleic acids in the sampleare not subjected to a purification technique that involves physicallyor chemically separating nucleic acids from other components in thecells that originally contained the nucleic acids. In some embodiments,“without purification” means not performing any step or steps thatremove a percentage of the non-nucleic acid components of the samplegreater than or equal to 1%, 2%, 3%, 4%, 5%, 10%, 20% or more prior tocontacting the sample with a nucleic acid amplification reagent.

In certain embodiments, the collected buccal samples may be directlycontacted with nucleic acid amplification reagents without anyintervening steps. This may be achieved by, for example, placing nucleicacid amplification reagents in a reaction vessel, using the cap of thevessel to collect a buccal sample, immediately inserting the cap intothe reaction vessel, and then bringing the nucleic acid amplificationreagents into contact with the buccal sample (e.g., by flicking thereaction vessel, inverting the reaction vessel, shaking the reactionvessel, vortexing the reaction vessel, etc.). Similarly, when using aswab to collect buccal samples, the swab may be dipped directly into thenucleic acid amplification reagents.

In certain embodiments, the collected swab samples may be subjected toan intervening step before being contacted with nucleic acidamplification reagents. For example, as discussed above, in certainsituations it may be advantageous to divide a swab sample into aliquotsso that more than one test may be performed for the same sample source.In some embodiments, a swab sample may be diluted in a vessel other thanthe reaction vessel (e.g., by mixing the swab sample with a buffer) andoptionally aliquoted before contacting the nucleic acid amplificationreagents in the reaction vessel. Those skilled in the art will recognizethat other similar intervening steps could be introduced into a methodof the present disclosure without deviating from the scope of variousembodiments of the present invention.

In various embodiments, template nucleic acids from the buccal samplemay be amplified using polymerase chain reaction (PCR) or reversetranscription PCR (RT-PCR); however, as noted previously, the skilledartisan will understand that numerous methods are known in the art foramplification of nucleic acids, and that these methods may be usedeither in place of, or together with, PCR or RT-PCR. For example,without limitation, other amplification methods employ ligase chainreaction (LCR), transcription-based amplification system (TAS), nucleicacid sequence based amplification (NASBA) reaction, self-sustainedsequence replication (3SR), strand displacement amplification (SDA)reaction, boomerang DNA amplification (BDA), Q-beta replication,isothermal nucleic acid sequence based amplification, etc. In general,nucleic acid amplification methods, such as PCR, RT-PCR, isothermalmethods, rolling circle methods, etc., are well known to the skilledartisan. See, e.g., Saiki, “Amplification of Genomic DNA” in PCRProtocols, Innis et al. (1990). Eds. Academic Press, San Diego, Calif.pp 13-20; Wharam et al. (2001). Nucleic Acids Res. 29(11): E54-E54;Hafner et al. (2001). Biotechniques. 30(4): 852-6, 858, 860 passim.

In certain embodiments, the nucleic acid amplification reagents that areinvolved in each of these amplification methods contain DNA polymeraseat a concentration higher than typically used in PCR reactions (1.0U/reaction). In various embodiments, the DNA polymerase concentration is2.0 U/reaction. The nucleic acid amplification reagents that areinvolved in each of these amplification methods (e.g., enzymes, primers,probes, buffers, etc.) may vary but are also well known in the art andreadily available from commercial sources (e.g., see catalogues fromInvitrogen, Biotools, New England Biolabs, Bio-Rad, QIAGEN,Sigma-Aldrich, Agilent Technologies, R&D Systems, etc.). It will also beappreciated that the specific primers and/or probes that are used in anygiven method will depend on the template nucleic acid and the targetsequence that is being amplified and that those skilled in the art mayreadily design and make suitable primers and/or probes for differenttemplate nucleic acids and target sequences. Primers and probes may alsobe prepared by commercial suppliers (e.g., Integrated DNA Technologies).

In another embodiment, the nucleic acid amplification reagents containprimer and probe concentrations higher than typically used in PCRreactions (0.1-0.2 μM). In some embodiments, the primer concentration is0.5 μM and the probe concentration is 0.7 μM.

In another embodiment, the nucleic acid amplification reagents containboth DNA polymerase, primer, and probe concentrations higher thantypically used in PCR reactions. In some embodiments, the DNA polymeraseprimer concentration is 2.0 U/reaction; the primer concentration is 0.5μM; and the probe concentration is 0.7 μM.

PCR is a technique for making many copies of a specific target sequencewithin a template DNA. The reaction consists of multiple amplificationcycles and is initiated using a pair of primer oligonucleotides thathybridize to the 5′ and 3′ ends of the target sequence. Theamplification cycle includes an initial denaturation and typically up to50 cycles of hybridization, strand elongation (or extension), and strandseparation (denaturation). The hybridization and extension steps may becombined into a single step. In each cycle of the reaction, the targetsequence between the primers is copied. Primers may hybridize to thecopied DNA amplicons as well as the original template DNA, so the totalnumber of copies increases exponentially with time/PCR cycle number. Insome embodiments, PCR may be performed according to methods described inWhelan et al. (1995). Journal of Clinical Microbiology. 33(3):556-561.Briefly, the nucleic acid amplification reagents (PCR reaction mixture)include two specific primers per target sequence, dNTPs, a DNApolymerase (e.g., approximately 0.25 U of the enzyme Taq polymerase),and a buffer (e.g., 1×PCR Buffer. The amplification reaction itself isperformed using a thermal cycler. Cycling parameters may be varied,depending on, for example, the melting temperatures of the primers orthe length of the target sequence(s) to be extended. As mentionedpreviously, the skilled artisan is capable of designing and preparingprimers that are appropriate for amplifying a target sequence. Thelength of the amplification primers for use in the present methodsdepends on several factors including the level of nucleotide sequenceidentity between the primers and complementary regions of the templatenucleic acid and also the temperature at which the primers arehybridized to the template nucleic acid. The considerations necessary todetermine a preferred length for an amplification primer of a particularsequence identity are well-known to a person of ordinary skill in theart and include considerations described herein. For example, the lengthand sequence of a primer may relate to its desired hybridizationspecificity or selectivity.

In certain embodiments, the nucleic acid amplification reaction isperformed within 120 minutes of contacting the buccal sample with thenucleic acid amplification reagents. In some embodiments, the nucleicacid amplification reaction is performed even sooner, e.g., within 60,30, 15, 10, 5 or even 1 minute(s) of contacting the swab sample with thenucleic acid amplification reagents.

In certain embodiments, the nucleic acid amplification reactioncomprises an initial heat denaturation step of 15 minutes or less. Insome embodiments, the initial heat denaturation step is shorter, e.g., 5minutes or less, 3 minutes or less, or 1 minute or less. In certainembodiments, the initial heat denaturation step is performed at atemperature in the range of about 85° C. to about 99° C., e.g., about93° C. to about 97° C., about 93° C. to about 95° C., or about 95° C. toabout 97° C., etc. In some embodiments, the initial heat denaturationstep is performed at about 95° C.

Detection of Nucleic Acids

The presence of amplified target sequences or amplicons may be detectedby any of a variety of well-known methods. For example, in someembodiments electrophoresis may be used (e.g., gel electrophoresis orcapillary electrophoresis). Amplicons may also be subjected todifferential methods of detection, for example, methods that involve theselective detection of variant sequences (e.g., detection of singlenucleotide polymorphisms or SNPs using sequence specific probes). Insome embodiments, amplicons are detected by real-time PCR.

Real-time PCR or end-point PCR may be performed using probes incombination with a suitable amplification/analyzer such as the SpartanDX-12 desktop DNA analyzer, which is a low-throughput PCR system withfluorescent detection capabilities. Briefly, probes specific for theamplified target sequence (e.g. molecular beacons, TaqMan probes) areincluded in the PCR amplification reaction. For example, molecularbeacons contain a loop region complementary to the target sequence ofinterest and two self-complementary stem sequences at the 5′ and 3′ end.This configuration enables molecular beacon probes to form hairpinstructures in the absence of a target complementary to the loop. Areporter dye is positioned at the 5′ end and a quencher dye at the 3′end. When the probes are in the hairpin configuration, the fluorophoreand quencher are positioned in close proximity and contact quenchingoccurs. During PCR, the fluorescently labeled probes hybridize to theirrespective target sequences; the hairpin structure is lost, resulting inseparation of the fluorophore and quencher and generation of afluorescent signal. In another example, TaqMan probes contain a reporterdye at the 5′ end and a quencher dye at the 3′ end. During PCR, thefluorescent labeled TaqMan probes hybridize to their respective targetsequences; the 5′ nuclease activity of the DNA polymerase (e.g., Taqpolymerase) cleaves the reporter dye from the probe and a fluorescentsignal is generated. When probes that hybridize to different targetsequences are used, these are typically conjugated with a differentfluorescent reporter dye. In this way, more than one target sequence maybe assayed for in the same reaction vessel. The increase in fluorescencesignal is detected only if the target sequence is complementary to theprobe and is amplified during PCR. A mismatch between probe and targetsequences greatly reduces the efficiency of probe hybridization andcleavage.

The Spartan DX-12 has the capability to measure fluorescence at thebeginning and end of the PCR thermal cycling, providing convenient“end-point” detection of amplicon accumulation.

Kits

The present disclosure also provides a kit comprising a reaction vessel,a cap for the reaction vessel, and a means for holding the cap, whereinthe cap comprises a tip that is capable of protruding into and matingwith the reaction vessel and an end opposing the tip that is capable ofmating with the means for holding the cap.

In some embodiments, the kit further comprises nucleic acidamplification reagents. In some embodiments, the cap is mated with thereaction vessel and the nucleic acid amplification reagents are locatedwithin the reaction vessel. In some embodiments, the reaction vessel isempty and the nucleic acid amplification reagents are located within oneor more containers that form part of the kit.

In some embodiments, the cap is mated with the reaction vessel. In someembodiments, the cap is mated with the means for holding the cap. Insome embodiments, the reaction vessel and cap are as shown in FIGS. 1-4.In some embodiments, the means for holding the cap is as shown in FIG.5-6.

In some embodiments, the kit further comprises a means for controlling anucleic acid amplification reaction in the reaction vessel. In someembodiments, the means for controlling a nucleic acid amplificationreaction in the reaction vessel comprises a thermal cycler with a wellfor receiving the reaction vessel. It will be appreciated that anythermal cycler may be used for this purpose.

In some embodiments, the kit further comprises a means for determiningwhether an amplification product has been produced as a result of thenucleic acid amplification reaction. In some embodiments, the means fordetermining whether an amplification product has been produced as aresult of the nucleic acid amplification reaction comprises a device formeasuring fluorescence in the reaction vessel while the reaction vesselis located within the means for controlling a nucleic acid amplificationreaction in the reaction vessel.

In some embodiments the components of the kit operate as follows. First,a swab sample is collected by contacting the cap of the reaction vessel(e.g., a Spartan DX-12 tube) with the sample source, then the cap isdirectly contacted with nucleic acid amplification reagents (e.g.,primers, dNTPs, DNA polymerase and buffer) that have been loaded intothe reaction vessel. The tube is then placed in the thermal cycler(e.g., of a Spartan DX-12 instrument) where a nucleic acid amplificationreaction takes place as a result of thermal cycling. The amplificationproducts are detected using a system that is capable of detectingfluorescence within the reaction vessels when loaded within the thermalcycler (e.g., the detection components of the Spartan DX-12 instrument).Finally, the fluorescent results are manually or automatically analyzedto determine (and optionally quantify) whether an amplification producthas been produced as a result of the nucleic acid amplification reaction(e.g., with the Spartan Analyzer Software or with Microsoft™ Excel™).

EXAMPLES

The following examples serve to illustrate the present invention. Theseexamples are in no way intended to limit the scope of the invention.

Example 1 Effect of the Amount of Taq DNA Polymerase on PCR Performancewhen Amplifying from Buccal Samples

FIGS. 7 and 8 show graphs demonstrating effects of concentration of TaqDNA polymerase has on the Endpoint Florescence (EPF) of buccal samplesamplified by a FAM probe and a CalFluor 610 probe in accordance withembodiments of the present invention. Three buccal swabs were collectedper individual by the same two technicians. Subjects were asked to rinsetheir mouths once with water prior to sample collection. The technicianscollected buccal samples using the cap of a Spartan tube (SpartanBioscience, Ottawa, Canada) connected to a cap holder device (Spartanswab) (Spartan Bioscience, Ottawa, Canada). The part of the cap whichfits inside the Spartan tube was swiped on the inside of each subject'scheek (one swipe up, one swipe down, repeated 3 to 4 times). The cap wasthen inserted into a tube containing PCR master mix, and the tube wastapped gently to ensure contact between the master mix and the part ofthe cap containing the buccal sample. Each of the three samplescollected from an individual were added to PCR master mix containingeither 1 U, 2 U or 3 U GoTaq polymerase (Promega). The procedure wasperformed multiple times on a total of six individuals, such that 18reactions were performed at each amount of GoTaq.

PCR primers were designed to amplify a 91 base pair amplicon from thehuman CYP 2C19 gene. The forward primer sequence was 5′-TgC AAT AAT TTTCCC ACT ATC ATT g-3′ (SEQ ID NO:1) and the reverse primer sequence was5′-CCA AAA TAT CAC TTT CCA TAA AAg CA-3′ (SEQ ID NO:2). Primers weremanufactured by Integrated DNA Technologies. The molecular beacon probesequence for the wild-type gene was5′-cgcagATTTCCC[G]GGAACCCctgcg-3′(SEQ ID NO:3). The probe was labeled atthe 5′ end with FAM and at the 3′ end with Black Hole Quencher-1 (BHQ1).The square brackets indicate the location of the wild-type singlenucleotide polymorphism (SNP). The stem portion of the molecular beaconis shown in lower case and the loop portion is shown in upper case. Themolecular beacon probe sequence for the mutant gene was5′-cgcagTTATTTCCC[A]GGAACCCctgcg-3′ (SEQ ID NO:4). The probe was labeledat the 5′ end with CalFluor 610 (CF610) and at the 3′ end with BlackHole Quencher-2 (BHQ2). The brackets indicate the location of the mutantSNP. The stem portion of the molecular beacon is shown in lower case andthe loop portion is shown in upper case. Probes were manufactured byBiosearch Technologies. All individuals included in the study were knownto carry both a wild-type and a mutant allele and are therefore expectedto be successful for PCR and generate a fluorescent signal for bothprobes.

The PCR master mix contained final concentrations of the followingcomponents:

-   -   Colorless PCR buffer (containing final 1.5 mM magnesium        chloride) (Promega, Cat. No. M792A): 1×    -   Additional magnesium chloride (Biotools): 2.5 mM    -   dNTPs (Invitrogen, Cat. No. 55082, 55083, 55084, 55085): 0.2 mM    -   Forward primer: 0.5 μM    -   Reverse primer: 0.5 μM    -   FAM probe: 0.7 μM    -   CalFluor 610 probe: 0.7 μM    -   GoTaq DNA polymerase (Promega, Cat. No. M400): 1 Unit/2 Units/3        Units

The PCR reaction volume was 20 μl. Positive reactions contained buccalsamples from individuals as detailed above. Negative control samplescontained no buccal material. A total of 18 positive reactions and 5-6negative control reactions were performed at each condition (1 U, 2 U, 3U Taq) were performed. PCR was performed using the Spartan DX-12instrument (Spartan Bioscience) in Spartan DX-12 tubes (SpartanBioscience).

The PCR program had the following steps:

1) Initial denaturation: 95° C. for 5 min for 1 cycle

2) Cycling denaturation: 95° C. for 1 sec for 50 cycles

3) Cycling hybridization/extension: 45° C. for 20 sec for 50 cycles

Results were analyzed using Microsoft™ Excel™. Endpoint fluorescence(EPF) was calculated for the FAM and CalFluor610 channels as thedifference between fluorescence levels at the first and last cycles ofthe PCR.

As shown in FIGS. 7 and 8, the final EPF was higher for samplescontaining 2 U or 3 U of Taq DNA polymerase than for samples containing1 U of Taq DNA polymerase for both the FAM channel and the CalFluor 610channel. Specifically, the average and standard deviation (SD) EPFvalues were as follows:

FAM channel EPF CalFluor 610 channel EPF Average SD Average SD Negativecontrol-1 U 4267.2 3324.6 6769.2 3028.4 Positive-1 U 26364.4 10567.355509.9 18569.8 Negative control-2 U 6608.5 2167.5 8844.7 2383.6Positive-2 U 46167.5 18522.6 84967.5 23500.3 Negative control-3 U 8862.51780.8 8741.2 2151.3 Positive-3 U 45972.8 13475.2 103321.2 25401.1

The results demonstrate that the use of Taq DNA polymerase at amountsgreater than that typically employed in PCR reactions (1 U/reaction)allows for superior discrimination between positive and negative sampleswhen amplifying directly from buccal samples without purification. Thisis especially important considering the wide range of human DNA inbuccal and saliva samples.

Example 2 Effect of Primer and Probe Concentrations on PCR Performancewhen Amplifying from Buccal Samples

FIGS. 9 and 10 show graphs demonstrating effects of concentration ofprimer and probe on Endpoint Florescence (EPF) of buccal samplesamplified by a FAM probe and by a CalFluor 610 probe in accordance withembodiments of the present invention. Four buccal swabs per individualwere collected from four individuals by the same two technicians in asingle session. Subjects were asked to rinse their mouths once withwater prior to sample collection. The technicians collected buccalsamples as described in Example 1. The cap was then inserted into a tubecontaining PCR master mix, and the tube was tapped gently to ensurecontact of the liquid with the part of the cap containing the buccalsample. One of each of the four samples collected from an individual wasadded to PCR master mix containing the following final concentrations ofprimers and probes:

[Primer] (μM) [Probe] (μM) 0.7 0.9 0.5 0.7 0.3 0.4 0.1 0.1

PCR primers and probes were designed to amplify a 91 base pair ampliconfrom the human CYP 2C19 gene. The primers and probes used were asdescribed in Example 1. All individuals included in the study were knownto carry both a wild-type and a mutant allele and are therefore expectedto be successful for PCR and generate a fluorescent signal for bothprobes.

The PCR master mix contained final concentrations of the followingcomponents:

-   -   Colorless PCR buffer (containing final 1.5 mM magnesium        chloride) (Promega, Cat. No. M792A): 1×    -   Additional magnesium chloride (Biotools): 2.5 mM    -   dNTPs (Invitrogen, Cat. No. 55082, 55083, 55084, 55085): 0.125        mM    -   Forward primer: 0.1-0.7 μM (see table above)    -   Reverse primer: 0.1-0.7 μM (see table above)    -   FAM probe: 0.1-0.9 μM (see table above)    -   CalFluor 610 probe: 0.1-0.9 μM (see table above)    -   GoTaq DNA polymerase (Promega, Cat. No. M400): 2 Units

The PCR reaction volume was 20 μl. Positive reactions contained buccalsamples from individuals as detailed above. Negative control samplescontained no buccal material. A total of 3-4 negative control reactionsand 4 positive reactions were performed for each primer/probeconcentration. PCR was performed using the Spartan DX-12 instrument(Spartan Bioscience, Ottawa, Canada) in Spartan DX-12 tubes (SpartanBioscience, Ottawa, Canada).

The PCR program had the following steps:

1) Initial denaturation: 95° C. for 5 min for 1 cycle

2) Cycling denaturation: 95° C. for 5 sec for 50 cycles

3) Cycling hybridization/extension: 55° C. for 20 sec for 50 cycles

Results were analyzed using Microsoft™ Excel™. Endpoint fluorescence(EPF) was calculated for the FAM and CalFluor610 channels as thedifference between fluorescence levels at the first and last cycles ofthe PCR.

As demonstrated in FIGS. 9 and 10, increasing concentration of primersand probes to levels greater than those typically employed in standardPCR reactions (˜0.1-0.2 μM) resulted in higher final EPF values andgenerally lower relative standard deviations for both the FAM channeland the CalFluor 610 channel. Specifically, average and standarddeviation (SD) EPF values were as follows:

CalFluor 610 FAM channel EPF channel EPF Average SD Average SD Negativecontrol 1476.1 1341.2 1222.9 294.1 0.1 μM primer/0.1 μM probe Positive1070.0 1981.6 1859.8 1595.2 0.1 μM primer/0.1 μM probe Negative control1769.2 878.1 2142.4 950.8 0.3 μM primer/0.4 μM probe Positive 15743.02900.6 24386.2 1737.6 0.3 μM primer/0.4 μM probe Negative control 3425.9907.5 4971.2 1526.1 0.5 μM primer/0.7 μM probe Positive 41231.7 6753.851208.9 10244.6 0.5 μM primer/0.7 μM probe Negative control 4940.41878.5 5534.5 1850.1 0.7 μM primer/0.9 μM probe Positive 54958.5 2841.763985.8 4657.9 0.7 μM primer/0.9 μM probe

The results demonstrate that the use of primers and probes atconcentrations higher than those typically employed in PCR reactions(0.1-0.2 μM) allows for superior discrimination between positive andnegative samples when amplifying directly from buccal samples withoutpurification. This is especially important considering the wide range ofhuman DNA in buccal and saliva samples.

Example 3 Combined Effect of Primer Concentration, Probe Concentration,and the Amount of Taq DNA Polymerase on PCR Performance when Amplifyingfrom Buccal Samples

FIGS. 11 and 12 show graphs demonstrating combined effects ofconcentration of Taq DNA polymerase, primer, and probe on EndpointFlorescence (EPF) of buccal samples amplified by a FAM probe and aCalFluor 610 probe, respectively, in accordance with embodiments of thepresent invention. Six buccal swabs per individual were collected fromthree individuals by the same two technicians in a single session.Subjects were asked to rinse their mouths once with water prior tosample collection. The technicians collected buccal samples as describedin Example 1. The cap was then inserted into a tube containing PCRmaster mix, and the tube was tapped gently to ensure contact of theliquid with the part of the cap containing the buccal sample. The buccalsamples collected from an individual were added to one of three PCRmaster mixes, containing the following final amount of Taq DNApolymerase and concentrations of primers and probes:

Taq DNA [Primer] (μM) [Probe] (μM) polymerase (Units) 0.2 0.2 1.0 0.50.7 1.0 0.5 0.7 2.0

PCR primers and probes were designed to amplify a 91 base pair ampliconfrom the human CYP 2C19 gene. The primers and probes used were asdescribed in Example 1. All individuals included in the study were knownto carry both a wild-type and a mutant allele and are therefore expectedto be successful for PCR and generate a fluorescent signal for bothprobes.

The PCR master mix contained final concentrations of the followingcomponents:

-   -   Colorless PCR buffer (containing final 1.5 mM magnesium        chloride) (Promega, Cat. No. M792A): 1×    -   Additional magnesium chloride (Biotools): 2.5 mM    -   dNTPs (Invitrogen, Cat. No. 55082, 55083, 55084, 55085): 0.2 mM    -   Forward primer: 0.2-0.5 μM (see table above)    -   Reverse primer: 0.2-0.5 μM (see table above)    -   FAM probe: 0.2-0.7 μM (see table above)    -   CalFluor 610 probe: 0.2-0.7 μM (see table above)    -   GoTaq DNA polymerase (Promega, Cat. No. M400): 1 or 2 Units (see        table above)

The PCR reaction volume was 20 μl. Positive reactions contained buccalsamples from individuals as detailed above. Negative control samplescontained no buccal material. A total of 4 negative controls and 6positive reactions were performed for each PCR master mix (see tableabove). PCR was performed using the Spartan DX-12 instrument (SpartanBioscience) in Spartan DX-12 tubes (Spartan Bioscience).

The PCR program had the following steps:

1) Initial denaturation: 95° C. for 5 min for 1 cycle

2) Cycling denaturation: 95° C. for 1 sec for 50 cycles

3) Cycling hybridization/extension: 45° C. for 20 sec for 50 cycles

Results were analyzed using Microsoft™ Excel™. Endpoint fluorescence(EPF) was calculated for the FAM and CalFluor610 channels as thedifference between fluorescence levels at the first and last cycles ofthe PCR.

As illustrated in FIGS. 11 and 12, in comparison to samples containing 1U of polymerase, 0.2 μM primers and 0.2 μM probes, the final EPF washigher for samples containing 1 U of polymerase, 0.5 μM primers and 0.7μM probes and the EPF for samples containing 2 U of polymerase, 0.5 μMprimers and 0.7 μM probes was increased further for both the FAM channeland the CalFluor 610 channel. These findings demonstrate, for example,there is an additive effect of increased primer/probe concentration andincreased amount of Taq. Specifically, the average and standarddeviation (SD) EPF values were as follows:

FAM channel CalFluor 610 EPF channel EPF Average SD Average SD Negativecontrol 1341.9 1101.9 2268.9 1535.5 0.2 μM primer/0.2 μM probe/ 1 U TaqPositive 8052.9 2536.6 21061.7 4119.4 0.2 μM primer/0.2 μM probe/ 1 UTaq Negative control 5040.8 2040.6 6402.5 1726.1 0.5 μM primer/0.7 μMprobe/ 1 U Taq Positive 19645.5 5919.6 41561.7 9964.2 0.5 μM primer/0.7μM probe/ 1 U Taq Negative control 6411.6 2709.3 8194.5 3572.5 0.5 μMprimer/0.7 μM probe/ 2 U Taq Positive 6088.5 11486.1 82200.1 27939.4 0.5μM primer/0.7 μM probe/ 2 U Taq

The results demonstrate that the combination of Taq DNA polymerase andprimer/probe concentrations higher than those typically employed in PCRreactions produce a synergistic effect that is superior to eithercondition alone. This allows for superior discrimination betweenpositive and negative samples when amplifying directly from buccalsamples without purification. This is especially important consideringthe wide range in the amounts of human DNA found in buccal and salivasamples.

Example 4 PCR Success Rate for Buccal Samples

Buccal samples were collected using three different methods of swabbing.Subjects were asked not to eat or drink anything but water for 5 minprior to sample collection. Subjects were asked to rinse their mouthsonce with water prior to sample collection

First, 31 individuals swiped the inside of their cheek a single timeusing the EasySwab™ (TrimGen, Cat. No. ES-100). The individuals thendipped and removed the end of the foam tip swab into a 75 μl solution of1.33×PCR buffer (Biotools), 3.33 mM Magnesium Chloride (Biotools), and0.167 mM dNTPs (Invitrogen). After the swab was removed, 15 μl wasaliquoted into a Spartan DX-12 tube and mixed with 5 μl of the followingsolution: 0.4 μM FAM-BHQ1plus probe (sequence described below), 2.4 μMCF10-BHQ2 probe (sequence described below), 2 μM PCR primers (sequencesdescribed below), and 1 unit of Taq polymerase (Biotools). The finalconcentrations are described below. The EasySwab™ was not simply dippedinto a Spartan DX-12 tube containing PCR master mix because theEasySwab™ is too large to easily fit inside the tube.

Second, 33 individuals swiped the inside of their cheek a total of sixtimes (one swipe up, one swipe down, repeated three times) using a capfrom the Spartan DX-12 tube (Spartan Bioscience, see FIG. 1). The capwas then inserted into a tube containing PCR master mix, and the tubewas tapped gently to ensure contact of the liquid with the part of thecap containing the sample.

Third, 19 individuals collected a sample by touching the cap of aSpartan DX-12 tube to their tongue once. The cap was then inserted intoa tube containing PCR master mix, and the tube was tapped gently toensure contact of the liquid with the part of the cap containing thesample.

PCR primers were designed to amplify a 91 base pair amplicon from thehuman CYP 2C19 gene. The forward primer sequence was 5′-TgC AAT AAT TTTCCC ACT ATC ATT g-3′ (SEQ ID NO:1) and the reverse primer sequence was5′-CCA AAA TAT CAC TTT CCA TAA AAg CA-3′ (SEQ ID NO:2). Primers weremanufactured by Integrated DNA Technologies. The TaqMan probe sequencefor the wild-type gene was ttatttccc[g]ggaacc (SEQ ID NO:3). The probewas labeled at the 5′ end with FAM and at the 3′ end with Black HoleQuencher-1 plus (BHQ1plus). The brackets indicate the location of thewild-type single nucleotide polymorphism (SNP). The TaqMan probesequence for the mutant gene was ttatttccc[a]ggaacc (SEQ ID NO:4). Theprobe was labeled at the 5′ end with CalFluor 610 (CF610) and at the 3′end with Black Hole Quencher-2 (BHQ2). The brackets indicate thelocation of the mutant SNP. Probes were manufactured by BiosearchTechnologies. All individuals are expected to be successful for PCR andgenerate a fluorescent signal for the FAM probe because all individualstested were known to carry at least one copy of the wild-type the targetallele and thus the reaction contains an internal positive control.

The PCR master mix contained final concentrations of the followingcomponents:

-   -   PCR buffer (Biotools): 1×    -   Magnesium chloride (Biotools): 2.5 mM    -   dNTPs (Invitrogen, Cat. No. 10297-018): 0.125 mM    -   Forward primer: 0.5 μM    -   Reverse primer: 0.5 μM    -   FAM probe: 0.1 μM    -   CalFluor 610 probe: 0.6 μM (included in some but not all        reactions—fluorescent data for the CalFluor 610 channel was not        used in the analysis)    -   DNA polymerase (Biotools): 1 Unit

The PCR reaction volume was 20 μl. PCR was performed using the SpartanDX-12 instrument (Spartan Bioscience) in Spartan DX-12 tubes (SpartanBioscience).

The PCR program had the following steps:

-   -   Initial denaturation: 95° C. for 15 min for 1 cycle    -   Cycling denaturation: 95° C. for 5 sec for 50 cycles    -   Cycling hybridization/extension: 50° C. for 10 sec for 50        cycles.

Results were analyzed using Microsoft™ Excel™. Endpoint fluorescence(EPF) was calculated for the FAM channel as the difference betweenfluorescence levels at the first and last cycles of the PCR. For eachswabbing method tested, a reaction was deemed to have failed if the EPFvalue in the FAM channel was smaller than or equal to the average EPF ofmultiple negative control reactions containing no nucleic acid template.

For the TrimGen swab, 28 out of 31 buccal samples generated a positivePCR result, with 3 failures (90.3% reliability rate). For the Spartancap swiped on the cheek, 30 out of 33 buccal samples generated apositive PCR result, with 3 failures (90.9% reliability rate). For theSpartan cap touched on the tongue, 17 out of 19 samples generated apositive PCR result, with 2 failures (89.5% reliability rate).

Methods that collect both more and less buccal material were alsoevaluated. To collect a large amount of buccal material, large-borepipette tips were used to perform scrapes on the inside of the cheek.The large-pore pipette tips were constructed by cutting off the bottom 1cm of a standard P1000 pipette tip. Material collected from multiplecheek scrapes was pooled and 3 μl of this material was added to aSpartan tube containing 20 μl of 1×PCR master mix. To gather a smallamount of buccal material, a fine pipette tip (10 μl) was touched to theside of the mouth once (contacting the buccal epithelium) and thentouched to 20 μl of 1×PCR master mix inside a Spartan tube. For bothmethods, the final concentrations of components of the PCR master mixwere as listed above. For both methods, 12 replicate Spartan tubes weretested. Results were analyzed as described above. For the large amountof material, 12 of 12 samples generated a positive PCR result (100%reliability rate). For the small amount of material, 7 of 12 samplesgenerated a positive PCR result (58.3% reliability rate).

Based on these results, it appears that buccal swabs enablesignificantly higher PCR reliability rates compared with directamplification from whole saliva samples (Ochert A S et al. (1994).Inhibitory effect of salivary fluids on PCR: potency and removal. GenomeResearch. 3: 365-368.). Another unexpected result was that thereliability rates were high across four of the five sample collectionmethods (TrimGen swab, Spartan cap swiped on cheek, Spartan cap touchedon tongue, Large bore pipette tip), despite the fact that these methodscollect significantly different amounts of buccal material. The factthat the reliability rate decreased for samples with a low amount ofmaterial (fine pipette tip touched to the corner of the mouth)demonstrates that there is a lower limit of buccal material required foroptimal reaction performance.

The range of buccal material collected by the Spartan cap methods weredetermined by spectrophotometric analysis of samples collected by the“Spartan DX-12 cap on cheek” and “Spartan DX-12 cap on tongue” methods.Specifically, a single individual self-collected 24 buccal samples usingthe “cap on cheek” technique and 12 buccal samples using the “cap ontongue” technique, as previously described. Absorbance of the samples at230 nm, 260 nm, and 280 nm was measured by loading a 1.5 μl aliquot ontothe pedestal of the NanoDrop™ spectrophotometer (Thermo Scientific). A1.5 μl aliquot of sterile water was used as the control sample. Eachbuccal sample was measured in duplicate.

Average absorbance readings for the two collection techniques were asfollows:

Sum (A230, A230 A260 A280 A260, A280) Cap on cheek 0.127 0.123 0.4470.697 (n = 24) Cap on tongue 0.067 0.049 0.188 0.304 (n = 12)

Since DNA concentration is correlated with the absorbance at 260 nm, itappears that the “cap on cheek” technique collects more than twice theamount of DNA as the “cap on tongue” technique. Nevertheless, the PCRsuccess rate of both methods was almost equivalent. This unexpectedfinding indicates the cap is capable of collecting a broad range ofacceptable amounts of material.

Example 5 Timing for Testing of Buccal Samples

Five individuals self-collected buccal samples by swiping the inside oftheir cheek a single time using the EasySwab™ (TrimGen, Cat. No.ES-100). The individuals then dipped the end of the foam tip swab into asolution of PCR buffer, Magnesium chloride, and dNTPs, as described inExample 4. The resulting solution was aliquoted equally into fiveSpartan DX-12 tubes (15 μl per tube), and mixed with 5 μl of thefollowing solution: 0.4 μM FAM-BHQ1plus probe, 2.4 μM CF10-BHQ2 probe, 2μM PCR primers, 1 unit of Taq polymerase (Biotools). This ensured thateach tube had the same amount of buccal material. As a control,duplicate reactions were run using 10 ng of purified genomic DNA. ThePCR primers and probes, master mix concentrations, reaction volumes, PCRcycling program, and method of analysis were the same as described inExample 4.

The tubes containing PCR master mix and buccal sample or purified DNAwere incubated at room temperature for different times before performingPCR:

-   -   Four tubes were incubated at room temperature for 0 min    -   Four tubes were incubated at room temperature for 15 min    -   Four tubes were incubated at room temperature for 30 min    -   Four tubes were incubated at room temperature for 60 min    -   Four tubes were incubated at room temperature for 90 min

Results showed that the final end-point fluorescence (fluorescence atthe last cycle minus fluorescence at the first cycle) was lower when thebuccal sample and PCR master mix were incubated for longer times at roomtemperature before performing PCR. Specifically, the average ratios offinal normalized end-point fluorescence achieved by reactions withbuccal sample versus purified DNA were as follows:

0 min 15 min 30 min 60 min 90 minGreen probe: (FAM) ttatttccc[g]ggaacc   (BHQ 1plus) (SEQ ID NO: 3)Swabs (n = 5) 1.00 0.92 0.70 0.63 0.19 Purified DNA 1.00 0.86 0.80 0.970.55 (n = 2) Ratio 1.00 1.07 0.88 0.65 0.35Red probe: (CF610) ttatttccc[a]ggaacc   (BHQ2) (SEQ ID NO: 4) Swabs (n =4) 1.00 0.89 0.67 0.46 0.20 Purified DNA 1.00 0.99 0.77 0.95 0.55 (n =2) Ratio 1.00 0.90 0.87 0.48 0.36

The end-point fluorescence values declined over time the longer thebuccal sample and PCR mixture was incubated at room temperature beforeperforming the reaction. The values also decline over time for thepurified DNA, but not nearly as much as for the buccal samples. Overall,the results indicate that the mixture of buccal sample and PCR mastermix should ideally be tested within 15-30 minutes of contacting thesample with the PCR master mix. The practical implications of theseresults are significant. By minimizing the time the PCR reaction is incontact with raw sample it is possible to mitigate the effects ofinhibitory substances in the sample and achieve enhanced reactionperformance

Example 6 PCR Success Rate for Buccal Samples with Different InitialHeating Times

Six individuals were instructed to swab the inside of their cheek usinga buccal swab (EasySwab™, TrimGen Corp., Cat. No. ES-100). Subjects wereasked not to eat or drink for 5 min prior to sample collection. Subjectswere asked to rinse their mouths once with water prior to samplecollection. For each subject, the buccal swab was dipped into a PCRmaster mix, as described in Examples 4 and 5. This master mix was thenmixed and aliquoted equally into five Spartan DX-12 tubes. This ensuredthat each tube had the same amount of buccal material.

The PCR primers and probes, master mix concentrations and reactionvolumes were the same as described in Example 4.

Results were analyzed using Microsoft™ Excel™. Endpoint fluorescence(EPF) was calculated as the difference between fluorescence levels atthe first and last cycles of the PCR. For each PCR program tested, areaction was deemed to have failed if the EPF value was smaller than orequal to the EPF of a negative control within the same run (containingno nucleic acid template) plus 2.5 times the standard deviation ofnegative control reactions from all runs.

The following five PCR programs were performed (one tube for eachprogram).

PCR Program #1:

1) Initial denaturation: 95° C. for 15 min for 1 cycle

2) Cycling denaturation: 95° C. for 5 sec for 50 cycles

3) Cycling hybridization/extension: 50° C. for 10 sec for 50 cycles

PCR Program #2:

1) Initial denaturation: 95° C. for 7 min for 1 cycle

2) Cycling denaturation: 95° C. for 5 sec for 50 cycles

3) Cycling hybridization/extension: 50° C. for 10 sec for 50 cycles

PCR Program #3:

1) Initial denaturation: 95° C. for 5 min for 1 cycle

2) Cycling denaturation: 95° C. for 5 sec for 50 cycles

3) Cycling hybridization/extension: 50° C. for 10 sec for 50 cycles

PCR Program #4:

1) Initial denaturation: 95° C. for 3 min for 1 cycle

2) Cycling denaturation: 95° C. for 5 sec for 50 cycles

3) Cycling hybridization/extension: 50° C. for 10 sec for 50 cycles

PCR Program #5:

1) Initial denaturation: 95° C. for 1 min for 1 cycle

2) Cycling denaturation: 95° C. for 5 sec for 50 cycles

3) Cycling hybridization/extension: 50° C. for 10 sec for 50 cycles

Results showed that an initial denaturation time of 1 min was sufficientfor successful amplification of 5 out of 6 buccal samples. In contrast,an initial denaturation time of 3, 5, 7, or minutes was sufficient forsuccessful amplification of 6 out of 6 buccal samples.

Results showed that an initial denaturation time of 1 min was sufficientfor successful amplification of 5 out of 6 buccal samples. In contrast,an initial denaturation time of 3, 5, 7, or minutes was sufficient forsuccessful amplification of 6 out of 6 buccal samples.

This result was unexpected given that the PCR success rate with wholesaliva samples was reported as 7 out of 10 samples after 5 minutes ofboiling followed by 1 min of cycling denaturation at 94° C. (Ochert A Set al. (1994). Inhibitory effect of salivary fluids on PCR: potency andremoval. Genome Research. 3: 365-368.)

Example 7 Higher Diagnostic Success Rate by Testing Two Swabs at theSame Time from the Same Individual

Buccal samples were collected from 25 individuals by the sametechnician. Subjects were asked not to eat or drink for 5 min prior tosample collection. The technician collected samples by taking the capfrom a Spartan DX-12 tube (Spartan Bioscience) and swiping the part ofthe cap containing the sample inside of the cheek (one swipe up, oneswipe down, repeated three times). The cap was then inserted into thetube, and the tube was tapped gently to ensure contact of the liquidwith the part of the cap containing the sample of the cap. Tubes werebriefly centrifuged to pull down the liquid into the bottom of the tube.Two samples were collected consecutively from each subject from roughlythe same area of the cheek using a separate cap for each sample.

The PCR primers and probes, master mix concentrations, reaction volumes,PCR cycling conditions, and method of analysis were the same asdescribed in Example 4.

For the first set of samples, the PCR success rate was 24 out of 25samples. For the second set of samples, the PCR success rate was 23 outof 25 samples.

The unexpected result was that failed samples were from differentindividuals for the first and second set of samples. For example, thesample that failed PCR in the first set of samples came from anindividual whose second sample resulted in a successful PCR. Similarly,the two individuals whose second samples failed PCR had first sampleswhich resulted in successful PCR. In other words, the diagnostic successrate from collecting and testing a single sample ranged from 92% to 96%,whereas the combined diagnostic success rate for two samples collectedat the same time was 100%. A clinically useful result only requires thatone of the samples be amplifiable by PCR.

The practical implications of these results are significant. Bycollecting and analyzing two or more buccal samples at the same timefrom the same individual, it is possible to mitigate the effects ofinhibitory substances in the sample and achieve a clinically usefulresult. This solves the reliability issue that has prevented saliva andbuccal samples from being routinely used for amplification withoutpurification.

Example 8 Cap Holder Device and Swabbing Procedure

To facilitate the use of the cap of a Spartan DX-12 tube to collect abuccal sample, one may construct an exemplary cap holder device usingthe following parts, shown in FIGS. 5 and 6:

-   -   One (1) Spartan DX-12 tube cap (Part #01004156), cap 501 in FIG.        5.    -   Three (3) 4 mm×150 mm drinking straws (Touch Industries, Part        #92-004). One straw, straw 502, cut to 118 mm in length with a        linear cut extending along the entire length of the straw. One        straw, straw 503, cut to 129 mm in length with a linear cut        extending along the entire length of the straw. One straw, straw        504, cut to 137 mm in length with a linear cut extending 10 mm        inward from one extremity of the straw.    -   One (1) 4 mm×150 mm drinking straw (Touch Industries, Part        #92-782). This straw, straw 505, is cut to 75 mm in length with        a linear cut extending along the entire length of the straw.        Straw 505 is used to make a cover for the Spartan DX-12 tube cap        505 and may have a closed end in accordance with various        embodiments of the present invention.

The assembly process is as follows:

-   -   Insert cap 501 into the end of straw 504 in the orientation        shown in FIG. 5. Cap 501 should be inserted into the portion of        straw 504 with a linear cut extending 10 mm inward from one        extremity. After assembly, the reference edge of straw 504, edge        514, should be 0.5±0.2 mm from the reference surface of cap 501,        surface 601, shown in FIG. 6.    -   Slide straw 502 over straw 504. After assembly, the reference        edges of straw 502 and straw 504, edges 512 and 514        respectively, shown in FIG. 5, should be aligned.    -   Insert straw 503 into the open end of straw 504 straw 503 comes        into contact with the reference surface of cap 501, surface 511,        shown in FIG. 5. After assembly, the reference edges of straws        503 and 504, edges 513 and 515, should be aligned.    -   Insert cover straw 505 over straw 504 so that the midpoint of        cover 505 is aligned with the exposed end of cap 501, as shown        in FIG. 5.    -   Fold the portion of straw 504 extending past the protruding end        of cap 501 over on itself so that it completely covers cap 501.

In this way, a swab has been manufactured out of a Spartan DX-12 tubecap and the drinking straws. While members 501-505 are described asstraws, these members may be composed of other tubular structures inaccordance with various embodiments of the present invention. Anexemplary swabbing procedure for using the manufactured swab is asfollows:

-   -   1) Have the subject rinse his or her mouth with water. The        subject may swallow or spit the rinse water.    -   2) Take one manufactured swab and one standard Spartan DX-12        reaction tube which has been pre-filled with nucleic acid        amplification reagents and capped. Remove the cap from the        Spartan DX-12 reaction tube and discard the cap.    -   3) Remove the cover (FIG. 5, Item “E”) from the swab and        discard.    -   4) Swab the inside of the subject's cheek by holding the shaft        of the swab and rubbing the cap from the swab against the cheek        up and down three times.    -   5) Insert the swab into the Spartan DX-12 reaction tube so that        the cap from the swab seals the tube. Place your thumb on the        end of the swab to ensure that there is sufficient pressure to        completely seat the cap.    -   6) While holding the swab vertically with the tube pointed        downward at an angle of approximately 30 degrees, gently tap the        tube three (3) times to mix the reagents.    -   7) Insert the sealed tube into the Spartan DX-12 instrument and        discard the shaft of the swab.

Example 9 Higher PCR Success Rate Using Dual HPLC Purified Probes andCap Holder Device

Subjects whose CYP450 2C19 *1 and *2 genotypes had been determinedpreviously were tested using three methods.

In the first method, PCR master mix was freshly mixed; aliquoted intoSpartan DX-12 tubes; capped with Spartan DX-12 tube caps; and left atroom temperature for 10 min. In parallel, 24 individuals swiped theinside of their cheek a total of six times (one swipe up, one swipedown, repeated three times) using a separate Spartan DX-12 tube cap.Subjects were asked not to eat or drink anything but water for 5 minprior to sample collection. Subjects were asked to rinse their mouthswith water prior to sample collection. The cap with the buccal samplewas then inserted into the tube containing PCR master mix, and the tubewas tapped gently to ensure contact of the liquid with the part of thecap containing the sample. The capped tube with buccal sample wasinserted into a Spartan DX-12 instrument 10 min after mixing the sample.

PCR primers were designed to amplify a 91 base pair amplicon from thehuman CYP 2C19 gene. The forward primer sequence was5′-TgCAATAATTTTCCCACTATCATTg-3′ (SEQ ID NO:1) and the reverse primersequence was 5′-CCAAAATATCACTTTCCATAAAAgCA-3′ (SEQ ID NO:2). Primerswere manufactured by Integrated DNA Technologies. The TaqMan probesequence for the wild-type gene was ttatttccc[g]ggaacc (SEQ ID NO:3).The probe was labeled at the 5′ end with FAM and at the 3′ end withBlack Hole Quencher-1 plus (BHQ1plus). The brackets indicate thelocation of the wild-type single nucleotide polymorphism (SNP). TheTaqMan probe sequence for the mutant gene was ttatttccc[a]ggaacc (SEQ IDNO:4). The probe was labeled at the 5′ end with CalFluor 610 (CF610) andat the 3′ end with Black Hole Quencher-2 (BHQ2). The brackets indicatethe location of the mutant SNP. Probes were manufactured by BiosearchTechnologies. All the individuals are expected to be successful for PCRand generate a fluorescent signal for the FAM probe because allindividuals tested were known to carry at least one copy of thewild-type target allele; thus, the reaction contains an internalpositive control.

The PCR master mix contained final concentrations of the followingcomponents:

-   -   PCR buffer (without Magnesium chloride) (Biotools): 1×    -   Magnesium chloride (Biotools): 2.5 mM    -   dNTPs (Invitrogen, Cat. No. 10297-018): 0.125 mM    -   Forward primer: 0.5 μM    -   Reverse primer: 0.5 μM    -   FAM probe: 0.1 μM    -   CalFluor 610 probe: 0.6 μM    -   DNA polymerase (Biotools): 1 Unit

The PCR reaction volume was 20 μl. PCR was performed using the SpartanDX-12 instrument (Spartan Bioscience).

The PCR program had the following steps:

1) Initial denaturation: 95° C. for 10 min for 1 cycle

2) Cycling denaturation: 95° C. for 5 sec for 50 cycles

3) Cycling hybridization/extension: 50° C. for 10 sec for 50 cycles

Endpoint fluorescence (EPF) was calculated for each sample bysubtracting the fluorescence value at the first cycle from thefluorescence value at the last cycle. A test result was called assuccessful if the EPF for the FAM channel was greater than 9,800arbitrary units. A test result was called as unsuccessful if the EPF forthe FAM channel was less than or equal to 9,800 arbitrary units. OverallPCR success rate was calculated as total number of successful resultsdivided by total number of results.

The same materials and methods were used in the second method, with thefollowing exceptions: (1) both probes were dual HPLC purified; (2) thefinal concentrations of the FAM probe and CF610 probe were 0.2 μM and0.4 μM, respectively; and (3) the temperature for cyclinghybridization/extension was 55° C.

The third method only differed from the second method in that the capholder device and swabbing method described in Example 8 were used tocollect the buccal sample from subjects.

The overall PCR success rates for the three methods were 91.7 percentfor the first method (24 subjects) 95.8 percent for the second method(24 subjects), and 97.9 percent for the third method (48 subjects).

Based on these results, PCR success rate may be improved by using dualHPLC purified probes.

The results in this example also indicate that PCR success rate may beimproved by using the cap holder device of Example 8 for the SpartanDX-12 tube cap to collect a concentration range of buccal material thatis compatible with direct amplification without purification. Theincrease in PCR success rate was found to be additive when combined withthe use of dual HPLC purified probes.

Example 10 Direct Amplification without Purification from Non-BuccalHuman Samples

Samples were collected from various body parts of one individual whoseCYP450 2C19 *1 and *2 genotypes had been determined previously. Thesamples were collected by rubbing a cap from the Spartan DX-12 tube onthe body part. Four samples each were collected from the palm, armpit,inside the folds of the pinna of the ear, and inside the nasal cavity.Prior to sample collection from the palm, the individual had beenclenching his fist and his palm was sweaty.

Within 1 min after sample collection, the cap was inserted into a tubecontaining PCR master mix, and the tube was tapped gently to ensurecontact of the liquid with the part of the cap containing the sample.The capped tube with sample was inserted into a Spartan DX-12 instrument10 min after mixing the sample.

PCR primers were designed to amplify a 91 base pair amplicon from thehuman CYP 2C19 gene. The forward primer sequence was5′-TgCAATAATTTTCCCACTATCATTg-3′ (SEQ ID NO:1) and the reverse primerssequence was 5′-CCAAAATATCACTTTCCATAAAAgCA-3′ (SEQ ID NO:2). Primerswere manufactured by Integrated DNA Technologies. Both primers were HPLCpurified. The TaqMan probe sequence for the wild-type gene wasttatttccc[g]ggaacc (SEQ ID NO:3). The probe was labeled at the 5′ endwith FAM and at the 3′ end with Black Hole Quencher-1 plus (BHQ1plus).The brackets indicate the location of the wild-type single nucleotidepolymorphism (SNP). The TaqMan probe sequence for the mutant gene wasttatttccc[a]ggaacc (SEQ ID NO:4). The probe was labeled at the 5′ endwith CalFluor 610 (CF610) and at the 3′ end with Black Hole Quencher-2plus (BHQ2plus). The brackets indicate the location of the mutant SNP.Probes were manufactured by Biosearch Technologies. Both probes weredual HPLC purified.

The PCR master mix contained final concentrations of the followingcomponents:

-   -   GoTaq master mix (containing final 1.5 mM magnesium chloride)        (Promega): 1×    -   Additional magnesium chloride (Biotools): 1.5 mM    -   Forward primer: 0.5 μM    -   Reverse primer: 0.5 μM    -   FAM probe: 0.2 μM    -   CalFluor 610 probe: 0.4 μM    -   GoTaq DNA polymerase (Promega): 1 Unit

The PCR reaction volume was 21 μl. PCR was performed using the SpartanDX-12 instrument (Spartan Bioscience).

The PCR program had the following steps:

1) Initial denaturation: 95° C. for 15 min for 1 cycle

2) Cycling denaturation: 95° C. for 5 sec for 50 cycles

3) Cycling hybridization/extension: 58° C. for 10 sec for 50 cycles

Results were analyzed using Microsoft™ Excel™. Endpoint fluorescence(EPF) was calculated for each sample by subtracting the fluorescencevalue at the first cycle from the fluorescence value at the last cycle.A test result was called as unsuccessful if the EPF was smaller than orequal to the average plus 2 times the standard deviation of EPF ofmultiple negative controls containing no nucleic acid template.

Results showed that the correct genotype was obtained in 3 out of 4 palmsamples, 1 out of 4 armpit samples, 2 out of 4 ear samples, and 3 out of4 nose samples. These results indicate that non-buccal samples may alsobe used for direct nucleic acid amplification without purification.

Example 11 Direct Amplification without Purification from Non-BuccalNon-Human Samples

Whole blood was collected from raw pork liver and DNA was purified usingthe Arrow DNA Purification Robot (NorDiag, Oslo, Norway) and the ArrowBlood-200 kit, according to the manufacturer's instructions. PurifiedDNA was quantified using the NanoDrop™ spectrophotometer (ThermoScientific). In parallel, a sample was collected from a fresh pork chopby rubbing a cap from the Spartan DX-12 tube on the raw meat Immediatelyafter sample collection, the cap was inserted into a tube containing PCRmaster mix, and the tube was tapped gently to ensure contact of theliquid with the part of the cap containing the sample. The capped tubewith sample was inserted into a Spartan DX-12 instrument after mixingthe sample.

PCR primers were designed to amplify a 130 base pair amplicon from theporcine Cytochrome B gene (Tanabe S et al. (2007). A quantitative PCRdetection method for pork, chicken, beef, mutton, and horseflesh infoods. Bioscience, Biotechnology, and Biochemistry. 71(12): 3131-3135.).The forward primer sequence was 5′-gttgcaaatcctaacaggcctg-3′ (SEQ IDNO:5) and the reverse primer sequence was5′-cgtttgcatgtagatagcgaataac-3′ (SEQ ID NO:6). Primers were manufacturedby Integrated DNA Technologies.

The PCR master mix contained final concentrations of the followingcomponents:

-   -   PCR Buffer (Biotools): 1×    -   dNTPs (Invitrogen): 0.125 mM    -   Magnesium chloride (Biotools): 2.5 mM    -   Forward primer: 0.5 μM    -   Reverse primer: 0.5 μM    -   SYBR Green (Invitrogen): 0.5×    -   Taq DNA polymerase (Biotools): 1 Unit

For the DNA purified from blood, the final amount of DNA in the reactionmixture was 21 ng. The PCR reaction volume was 20 μl. PCR was performedusing the Spartan DX-12 instrument (Spartan Bioscience).

The PCR program had the following steps:

1) Initial denaturation: 95° C. for 10 min for 1 cycle

2) Cycling denaturation: 95° C. for 30 sec for 50 cycles

3) Cycling hybridization/extension: 60° C. for 60 sec for 50 cycles

Amplicons were analyzed using gel electrophoresis. PCR generatedamplicons of the expected size for DNA purified from blood and samplescollected by rubbing the Spartan DX-12 cap on the raw pork chop.

This experiment demonstrates that swabs from non-human samples may beused for direct amplification without purification according to themethod described in this invention. Other suitable non-human samples mayinclude and are not limited to fish, shellfish, beef, chicken, and lamb.

Other Embodiments

Other embodiments will be apparent to those skilled in the art from aconsideration of the specification or practice of the inventiondisclosed herein. It is intended that the specification and examples beconsidered as exemplary only, with the true scope of the invention beingindicated by the following claims. The entire contents of any referencethat is referred to herein are hereby incorporated by reference.

1. A method comprising steps of: obtaining a swab sample comprisingnucleic acid from a source; and contacting the swab sample with anucleic acid amplification reagent without purification of the nucleicacid from the sample, wherein the nucleic acid amplification reagentcomprises at least one of a DNA polymerase concentration of at least 1.0U/reaction, a primer concentration of at least 0.2 μM, and a probeconcentration of at least 0.2 μM.
 2. The method of claim 1, wherein theDNA polymerase concentration is at least 2.0 U/reaction; the primerconcentration is at least 0.5 μM; and the probe concentration is 0.7 μMor greater.
 3. The method of claim 1 further comprising determiningwhether an amplification product was produced as a result of the nucleicacid amplification reaction.
 4. The method of claim 1, where the nucleicacid amplification reaction is performed within 120 minutes ofcontacting the swab sample with the nucleic acid amplification reagents.5. The method of claim 1, where the nucleic acid amplification reactionis performed within 60 minutes of contacting the swab sample with thenucleic acid amplification reagents.
 6. The method of claim 1, where thenucleic acid amplification reaction is performed within 30 minutes ofcontacting the swab sample with the nucleic acid amplification reagents.7. The method of claim 1, where the nucleic acid amplification reactionis performed within 15 minutes of contacting the swab sample with thenucleic acid amplification reagents.
 8. The method of claim 1, where thenucleic acid amplification reaction is performed within 10 minutes ofcontacting the swab sample with the nucleic acid amplification reagents.9. The method of claim 1, where the nucleic acid amplification reactionis performed within 5 minutes of contacting the swab sample with thenucleic acid amplification reagents.
 10. The method of claim 1, wherethe nucleic acid amplification reaction is performed within 1 minute ofcontacting the swab sample with the nucleic acid amplification reagents.11. The method of claim 1, where the performing a nucleic acidamplification reaction on the swab sample comprises an initial heatdenaturation step of 15 minutes or less.
 12. The method of claim 1,where the performing a nucleic acid amplification reaction on the swabsample comprises an initial heat denaturation step of 5 minutes or less.13. The method of claim 1, where the performing a nucleic acidamplification reaction on the swab sample comprises an initial heatdenaturation step of 3 minutes or less.
 14. The method of claim 1, wherethe performing a nucleic acid amplification reaction on the swab samplecomprises an initial heat denaturation step of 1 minute or less.
 15. Themethod of claim 1 further comprising collecting the swab sample.
 16. Themethod of claim 15, where the swab sample is collected from a mammal 17.The method of claim 16, where the mammal is a human.
 18. The method ofclaim 16, where the swab sample is collected from an open body cavity.19. The method of claim 16, where the swab sample is collected from abody surface.
 20. The method of claim 16, where the swab sample is abuccal sample.
 21. The method of claim 16, where the swab sample iscollected from the palm of a hand, inside the folds of the pinna of anear, an armpit, or inside a nasal cavity.
 22. The method of claim 15,where the swab sample is collected from a foodstuff.
 23. The method ofclaim 22, where the foodstuff is raw.
 24. The method of claim 22, wherethe foodstuff is a fruit, a vegetable, a meat, a fish, or a shellfish.25. The method of claim 24, where the meat is pork, beef, chicken orlamb.
 26. The method of claim 15, where the nucleic acid amplificationreaction is performed in a reaction vessel having a removable cap andthe cap is used to collect the swab sample.
 27. The method of claim 26,where a means for holding the cap is used when collecting the swabsample
 28. The method of claim 26, where the cap is contacted with theinside of a cheek.
 29. The method of claim 26, where the cap iscontacted with a tongue.
 30. The method of claim 26, where the cap iscontacted with the palm of a hand, the inside of the folds of the pinnaof an ear, an armpit, or the inside of a nasal cavity.
 31. The method ofclaim 26, where the cap is contacted with a foodstuff.
 32. The method ofclaim 31, where the foodstuff is raw.
 33. The method of claim 31, wherethe foodstuff is a fruit, a vegetable, a meat, a fish, or a shellfish.34. The method of claim 33, where the meat is pork, beef, chicken orlamb.
 35. The method of claim 15, where the swab sample is collectedfrom a source and the method is repeated with at least one other swabsample from the same source.
 36. The method of claim 1, where the swabsample is directly contacted with the nucleic acid amplificationreagents without any intervening steps.
 37. The method of claim 36,where the nucleic acid amplification reaction is performed in a reactionvessel having a removable cap and the cap is used to collect the swabsample.
 38. The method of claim 37, where reaction vessel comprises thenucleic acid amplification reagents and the cap is used to seal thereaction vessel after the swab sample has been collected.
 39. The methodof claim 36, where a means for holding the cap is used when collectingthe swab sample.
 40. A kit comprising: a reaction vessel; a cap for thereaction vessel; and a means for holding the cap, wherein the capcomprises a tip that is capable of protruding into and mating with thereaction vessel and an end opposing the tip that is capable of matingwith the means for holding the cap.
 41. The kit of claim 40 furthercomprising nucleic acid amplification reagents.
 42. The kit of claim 41,where the cap is mated with the reaction vessel and the nucleic acidamplification reagents are located within the reaction vessel.
 43. Thekit of claim 41, where the reaction vessel is empty and the nucleic acidamplification reagents are located within one or more containers thatform part of the kit.
 44. The kit of claim 43, where the cap is matedwith the reaction vessel.
 45. The kit of claim 43, where the cap ismated with the means for holding the cap.
 46. The kit of claim 40further comprising a means for controlling a nucleic acid amplificationreaction in the reaction vessel.
 47. The kit of claim 46, where themeans for controlling a nucleic acid amplification reaction in thereaction vessel comprises a thermal cycler with a well for receiving thereaction vessel.
 48. The kit of claim 46 further comprising a means fordetermining whether an amplification product has been produced as aresult of the nucleic acid amplification reaction.
 49. The kit of claim48, where the means for determining whether an amplification product hasbeen produced as a result of the nucleic acid amplification reactioncomprises a device for measuring fluorescence in the reaction vesselwhile the reaction vessel is located within the means for controlling anucleic acid amplification reaction in the reaction vessel.
 50. Themethod according to claim 1, wherein the nucleic acid amplificationreagent is contacted with the unpurified nucleic acid within a cappedcontainer, the cap of the container including a collection swab foracquiring the unpurified nucleic acid directly from a patient.
 51. Themethod according to claim 3, wherein contacting the nucleic acidamplification reagent with the unpurified nucleic acid comprisesagitating the container.
 52. The method according to claim 1 wherein thenucleic acid amplification reagent is exclusive of the Phusion™ HotStart II High Fidelity DNA polymerase enzyme.
 53. The method accordingto claim 1 wherein the nucleic acid amplification reagent is notmodified to resist inhibitors.
 54. A method comprising steps of:obtaining a swab sample comprising nucleic acid from a source; andcontacting the swab sample with a nucleic acid amplification reagentwith purification of the nucleic acid from the sample and without anyintervening steps, and where the nucleic acid amplification reaction isperformed within 60 minutes of contacting the swab sample with thenucleic acid amplification reagents.